Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines

ABSTRACT

This invention is directed to sequences that are involved in the molecular pathways of tumor suppression, tumor revision, apoptosis and/or virus resistance. The use of the compounds having these sequences, or encoded by them, in treating cancer, neurodegenerative diseases or viral diseases, and screening methods for compounds having such therapeutic properties are also encompassed.

The present invention relates to the identification of genes involved in the molecular pathways of tumour suppression, tumour reversion, apoptosis and/or resistance to viruses.

The present invention was made possible by the isolation of cDNA corresponding to messenger RNAs expressed or repressed during tumour suppression, tumour reversion and/or during the process of apoptosis.

In order to isolate the genes activated or inhibited during tumour reversion, overall screening for the expression of the genes in a malignant cell line (U937) and a derived cell line (US4) was carried out with suppression of the malignant phenotype. Comparison of the expressed genes (messenger RNAs expressed in both types of cell) made it possible to identify genes expressed differentially, that is to say expressed in one of the cells while they are not in the other (the genes may be activated or inhibited).

It is easily deduced therefrom that these genes are at least involved in the cancerization process, in one case by their absence, and in the other case by their presence.

For this differential study, the method used is the method described in 2000 by Brenner et al. (Proc. Natl. Acad. Sci. USA, 97 (4), 1665-70).

In order to produce a model, the inventors made the following hypotheses: if it were possible to select, from a tumour which is sensitive to the cytopathic effect of the parvovirus H-1, cells which are resistant, then this resistance could be due to a change in their malignant phenotype. It has been possible to demonstrate this for US4 cells selected from the U937 cancer cells. Unlike the U937 parental line, the US4 clones (but also US3 clones which will not be dealt with in the present invention) are resistant to the cytopathic effect of the parvovirus H-1.

At the molecular level, it has been possible to observe that this suppression of the malignant phenotype was accompanied by activation of the expression of the p21^(waf1) gene, this being independently of the expression of the p53 gene.

The approach to the problem according to the present invention made it possible to isolate sequences directly linked to several precise functions. Accordingly, unlike the random sequencing of ESTs, the sequences are sequences whose function is known since they are involved in the process of suppression of the malignant phenotype, of tumour reversion, of apoptosis and/or in resistance to viruses.

Tumour reversion is distinguishable from tumour suppression by the fact that it covers a broader domain than that of tumour suppressor genes. In other words, tumour reversion is achieved by the use of metabolic and/or molecular pathways which are not limited to the metabolic and molecular pathways in which the tumour suppressor genes are involved.

Thus, the present invention relates in particular to novel sequences and to the use of these sequences in the diagnostic field and for carrying out methods for screening test compounds. The invention also relates to methods for the detection and/or assay of the sequences of the invention or of their product(s) of expression in a biological sample.

The present invention first of all relates to an isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising:

-   -   a) SEQ ID No. 1 to SEQ ID No. 1020, preferably SEQ ID No. 72,     -   b) a nucleotide sequence of at least 15 consecutive nucleotides         of a sequence as defined in a),     -   c) a nucleotide sequence exhibiting a percentage identity of at         least 80%, after optimum alignment, with a sequence defined         in a) or b),     -   d) a nucleotide sequence which hybridizes under high stringency         conditions with a sequence defined in a) or b), and     -   e) a complementary nucleotide sequence where the RNA sequence         corresponding to a sequence as defined in a), b), c) or d).

The nucleotide sequence according to the invention defined in c) exhibits a percentage identity of at least 80% after optimum alignment with a sequence as defined in a) or b) above, preferably of at least 90%, more preferably of at least 98%.

In the context of the present invention, SEQ ID No. 72 is the preferred nucleotide sequence and corresponds to the TPT1 gene also called TCTP. This gene is involved in particular in the phenomenon of tumour reversion and has been the subject of more thorough experiments on the part of the inventors as indicated below.

The expressions nucleotide sequence, nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide and polynucleotide sequence, terms which will be used without distinction in the present description, are understood to mean a precise succession of nucleotides, modified or unmodified, which makes it possible to define a fragment or a region of a nucleic acid, containing, or otherwise, unnatural nucleotides, and being capable of corresponding at the same time to a double-stranded DNA, a single-stranded DNA and transcriptional products of the said DNAs. Thus, the nucleic sequences according to the invention also cover PNAs (Peptide Nucleic Acids), or the like.

The fragments of the nucleotide sequences of the invention comprise at least 15 consecutive nucleotides. Preferably, they comprise at least 20 consecutive nucleotides and still more preferably they comprise at least 30 consecutive nucleotides.

It should be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. It involves sequences which have been isolated and/or purified, that is to say that they were collected directly or indirectly, for example by copying, their environment having been at least partially modified. The nucleic acids obtained by chemical synthesis are understood to also be thereby designated.

The expression “percentage identity” between two nucleic acid or amino acid sequences for the purposes of the present invention is understood to mean a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, which is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed and over their entire length. The expression “best alignment” or “optimum alignment” is understood to mean the alignment for which the percentage identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, the said comparison being carried out per segment or per “comparison window” in order to identify and compare the local regions with sequence similarity. Optimum alignment of the sequences for comparison may be carried out, apart from manually, by means of the Smith and Waterman local homology algorithm (1981), by means of the Neddleman and Wunsch local homology algorithm (1970), by means of the Pearson and Lipman search for similarity methods (1988), by means of computer software packages using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.). In order to obtain optimum alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix. The PAM or PAM 250 matrices can also be used.

The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned optimally, it being possible for the nucleic acid or amino acid sequence to be compared to comprise additions or deletions in relation to the reference sequence for an optimum alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.

The expression nucleic sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with a reference sequence, is understood to mean the nucleic sequences exhibiting, in relation to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, alignment, chimeric fusion and/or substitution, in particular point substitution, and whose nucleic sequence exhibits at least 80%, preferably at least 90%, more preferably at least 98%, identity after optimum alignment with the reference nucleic sequence. Preferably, the specific or high stringency hybridization conditions will be such that they ensure at least 80%, preferably at least 90%, more preferably at least 98% identity after optimum alignment between one of the two sequences and the sequence complementary to the other.

Hybridization under high stringency conditions means that the temperature and ionic strength conditions are chosen so that they allow hybridization to be maintained between two complementary nucleic acid fragments. By way of illustration, high stringency conditions of the hybridization step for the purposes of defining the nucleotide sequences described above, will be advantageously the following.

The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution containing 0.15 M NaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10×Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization proper for 20 hours at a temperature dependent on the size of the probe (i.e.: 42° C., for a probe of size>100 nucleotides) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS, 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS. The final wash is performed in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe of size>100 nucleotides. The high stringency hybridization conditions described above for a polynucleotide of defined size may be adjusted by persons skilled in the art for larger- or smaller-size oligonucleotides, according to the teaching of Sambrook et al., 1989.

Among the nucleotide sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with the sequences according to the invention, preference is also given to the variant nucleic sequences of the sequences of the invention, or of fragments thereof, that is to say all the nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequences of the invention.

The expression “variant nucleotide sequence” is understood to mean any RNA or cDNA resulting from a mutation and/or variation of a splice site of the genomic DNA corresponding to the nucleotide sequences of the invention.

The subject of the present invention is also a polypeptide encoded by a nucleotide sequence in accordance with the invention.

The expression “polypeptide” is understood to mean, for the purposes of the present invention, either proteins or peptides.

According to a particular embodiment, the polypeptides in accordance with the invention comprise a polypeptide chosen from:

-   -   a) a polypeptide encoded by a nucleotide sequence in accordance         with the invention,     -   b) a polypeptide exhibiting at least 80% identity with a         polypeptide as defined in a),     -   c) a fragment of at least 5 amino acids of a polypeptide as         defined in a) orb),     -   d) a biologically active fragment of a polypeptide as defined in         a), b) or c), and     -   e) a modified polypeptide of a polypeptide as defined in a),         b), c) or d).

The percentage identity is understood to be evaluated after optimum alignment of the relevant sequences. The expression polypeptide whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98% after optimum alignment with a reference sequence, is understood to mean the polypeptides exhibiting certain modifications in relation to the reference polypeptide, such as in particular one or more deletions or truncations, an extension, a chimeric fusion and/or one or more substitutions.

Among the polypeptides whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90% and more preferably of at least 98%, after optimum alignment with a reference sequence such as a polypeptide in accordance with the present invention or with one of its fragments, preference is given to the variant polypeptides encoded by the variant nucleotide sequences as previously defined, in particular the polypeptides whose amino acid sequence exhibits at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one residue in relation to the polypeptide sequences of the invention or with one of their fragments.

The present invention also relates to a cell cloning and/or expression vector, characterized in that it comprises a nucleotide sequence according to the invention or that it encodes a polypeptide according to the invention. Such a vector may also contain the elements necessary for the expression and possibly for the secretion of the polypeptide in a host cell. Such a host cell is also a subject of the present invention.

The vectors comprising promoter and/or regulatory sequences also form part of the present invention. The said vectors preferably contain a promoter, signals for initiation and termination of translation, and appropriate regions for regulation of transcription. They should be able to be stably maintained in the cell and they may also possess particular signals so as to allow the secretion of the translated protein.

These different control signals are chosen according to the cellular host used. To this effect, the nucleic acid sequences according to the invention may be inserted into vectors which replicate autonomously in the chosen host or into integrating vectors of the chosen host.

Among the autonomously replicating systems, use is preferably made, according to the host cell, of the plasmid or viral type systems, it being possible for the viral vectors in particular to be adenoviruses (5), retroviruses, lentiviruses, poxviruses or herpesviruses (5a). Persons skilled in the art know the technologies which can be used for each of these systems.

When the integration of the sequence into the chromosomes of the host cell is desired, it is possible to use, for example, systems of the plasmid or viral type; such viruses are, for example, retroviruses (6) or AAVs (7).

Advantageously, the vectors in accordance with the invention contain a sequence allowing tissue-specific targeting and/or expression.

Among the nonviral vectors, preference is given to naked polynucleotides such as naked DNA or naked RNA according to the technique developed by the company VICAL, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) for expression in yeast, mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC) for expression in human cells.

Such vectors are prepared according to methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.

The invention additionally comprises the host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention and the transgenic animals, preferably the mammals, except humans, comprising one of the said transformed cells according to the invention. These animals may be used as models, for studying the aetiology of inflammatory and/or immune diseases, and in particular inflammatory diseases of the digestive tube, or for studying cancers.

Among the cells which can be used for the purposes of the present invention, there may be mentioned bacterial cells (8), but also yeast cells (9), as well as animal cells, in particular mammalian cell cultures (10), and in particular Chinese hamster ovary (CHO) cells. There may also be mentioned insect cells in which it is possible to use methods using for example baculoviruses (11). A preferred cellular host for the expression of the proteins of the invention consists of COS cells.

Among the mammals according to the invention, preference is given to animals such as rodents, in particular mice, rats or rabbits, expressing a polypeptide according to the invention.

These transgenic animals are obtained, for example, by homologous recombination on embryonic stem cells, transfer of these stem cells into embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of the said chimeras.

The transgenic animals according to the invention can thus overexpress the gene encoding the protein according to the invention, or their homologous gene, or may express the said gene into which a mutation is introduced. These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of a ubiquitous nature, or which is selective for a type of tissue, or after viral transcription.

The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can likewise serve as a model for analysis.

The transformed cells or mammals as described above may also be used as models in order to study the interactions between the polypeptides according to the invention and the chemical or protein compounds involved directly or indirectly in the activities of the polypeptides according to the invention, this being in order to study the different mechanisms and interactions involved.

They can in particular be used for the selection of products interacting with the polypeptides according to the invention, or their [lacuna] as a cofactor, or as an inhibitor, in particular competitive, or having an agonist or antagonist activity on the activity of the polypeptides according to the invention.

Preferably, the said transformed cells or transgenic animals are used as a model in particular for the selection of products which make it possible to control pathologies linked to abnormal expression of this gene.

The subject of the present invention is also a monoclonal or polyclonal antibody, a fragment of this antibody or a chimeric antibody capable of specifically recognizing a polypeptide in accordance with the present invention.

The specific monoclonal antibodies may be obtained according to the conventional hybridoma culture method well known to persons skilled in the art.

The antibodies according to the invention are for example humanized antibodies, Fab or F(ab′)² fragments. They may also be provided in the form of immunoconjugates or antibodies labelled so as to obtain a detectable and/or quantifiable signal.

The antibodies in accordance with the invention, but also the immunoconjugates, are therefore capable of specifically recognizing a polypeptide according to the present invention.

The specific polyclonal antibodies may be obtained from the serum of an animal immunized against the polypeptides of the invention, in particular produced by genetic recombination or by peptide synthesis, according to the customary procedures.

The importance of antibodies specifically recognizing the polypeptides, their variants or their immunogenic fragments, according to the invention,: should be noted in particular.

The subject of the invention is also the use of a nucleotide sequence in accordance with the present invention as a probe or a primer for the detection, identification, assay and/or amplification of nucleic acid sequences.

According to the invention, the nucleotide sequences which may be used as a probe or as a primer in methods for the detection, identification, assay and/or amplification of a nucleic acid sequence have a minimum size of 15 bases, preferably of 20 bases, or even better from 25 to 30 bases.

The probes and primers according to the invention may be directly or indirectly labelled with a radioactive or nonradioactive compound by methods well known to persons skilled in the art, so as to obtain a detectable and/or quantifiable signal.

The nonlabelled nucleic sequences according to the invention may be used directly as a probe or a primer.

The sequences are generally labelled in order to obtain sequences which can be used for numerous applications. The labelling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.

Among the radioactive isotopes used, there may be mentioned ³²P, ³³P, ³⁵S, ³H or ¹²⁵I. The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, colourings, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent and phosphorescent agents.

The nucleotide sequences according to the invention may thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (11a). This technique requires the choice of pairs of oligonucleotide primers flanking the fragment which has to be amplified. Reference may be made, for example, to the technique described in American Patent U.S. Pat. No. 4,683,202. The amplified fragments may be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion-exchange chromatography, and then sequenced. The specificity of the amplification may be checked, using as primers, the nucleotide sequences of the invention and, as templates, plasmids containing these sequences or the derived products of amplification. The amplified nucleotide fragments may be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid having a sequence complementary to that of the said amplified nucleotide fragments.

The invention also relates to the nucleic acids which can be obtained by amplification using primers according to the invention.

Other techniques for amplifying the target nucleic acid may be advantageously used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention. The expression PCR-like is understood to mean any of the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labelling systems were amplified, these techniques are of course known. In general, they involve the amplification of DNA by a polymerase, when the original sample is an RNA, a reverse transcription should be carried out beforehand. A great number of methods allowing this amplification currently exist, such as for example the SDA (Strand Displacement Amplification) technique (12), the TAS (Transcription-based Amplification System) technique described by (13), the 3SR (Self-Sustained Sequence Replication) technique described by (14), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by (15), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by (16), the RCR (Repair Chain Reaction) technique described by (17), the CPR (Cycling Probing Reaction) technique described by (18), the Q-beta-replicase amplification technique described by (19). Some of these techniques have since been improved.

In the case where the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to the use of an amplification reaction with the aid of the primers according to the invention or to the use of a method of detection with the aid of the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or probes used in the method of amplification or detection according to the invention.

The probe hybridization technique may be carried out in various ways (20). The method most generally used consists in immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well defined conditions, the immobilized target nucleic acid with the probe. After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, the fluorescence or the enzymatic activity linked to the probe).

According to another embodiment of the nucleic probes according to the invention, they may be used as capture probes. In this case, a probe, called “capture probe”, is immobilized on a support and serves to capture, through specific hybridization, the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by means of a second probe, called “detection probe”, labelled with an easily detectable element.

The nucleotide sequences according to the invention may moreover be of interest when they are used as antisense nucleotides, that is to say whose structure brings about, through hybridization with the target sequence, inhibition of the expression of the corresponding product. They may also be used as sense nucleotides which, through interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition, or an activation of this expression.

The subject of the invention is also the use of a nucleotide sequence according to the present invention for the production or the synthesis of a recombinant polypeptide.

The method of producing a polypeptide of the invention in recombinant form, which is itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultured under conditions allowing the expression of a recombinant polypeptide encoded by a nucleotide sequence according to the invention, and that the said recombinant polypeptide is recovered.

The recombinant polypeptides, characterized in that they can be obtained by the said method of production, also form part of the invention.

The recombinant polypeptides obtained as indicated above may be provided either in glycosylated or nonglycosylated form and may or may not have the natural tertiary structure.

The sequences of the recombinant polypeptides may also be modified in order to improve their solubility, in particular in aqueous solvents.

Such modifications are known to persons skilled in the art, such as for example the deletion of hydrophobic domains or the replacement of hydrophobic amino acids with hydrophilic amino acids.

These polypeptides may be produced from the nucleic acid sequences defined above, according to techniques for producing recombinant polypeptides known to persons skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.

An effective system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.

These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the transfected nucleotide sequence.

The methods used for purifying a recombinant polypeptide are known to persons skilled in the art. The recombinant polypeptide may be purified from cell lysates and extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity methods using specific monoclonal or polyclonal antibodies, and the like.

The polypeptides according to the present invention may also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the techniques using solid phases (21) or techniques using partial solid phases, by condensation of fragments or by conventional synthesis in solution.

The polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.

The subject of the present invention is also a DNA chip, characterized in that it contains at least one nucleotide sequence in accordance with the present invention.

Actually, the nucleotide sequences according to the invention which it is envisaged to use as probe or as primer for the detection, identification, assay and/or amplification of nucleic acid sequences may be covalently or noncovalently immobilized on a support, this support being a DNA chip or a high density filter.

The expression “DNA chip” or “high density filter” is understood to mean a support to which DNA sequences are attached, it being possible for each of them to be located by its geographic localization. These chips or filters differ mainly in their size, the support material, and possibly the number of sequences attached thereto.

In particular, it is possible to carry out a synthesis in situ by photochemical targeting or by ink jet targeting. Other techniques consist in carrying out a synthesis ex situ or in attaching the probes to the support of the DNA chip by ink jet, electronic or mechanical addressing. These different methods are well known to persons skilled in the art.

The subject of the invention is also a protein chip comprising a polypeptide or antibody according to the invention.

Such a protein chip allows the study of interactions between the polypeptides according to the invention and other proteins or chemical compounds and can thus be useful for screening compounds which interact with the polypeptides according to the invention.

It may also be possible to use the protein chips according to the invention for detecting the presence of antibodies directed against the polypeptides according to the invention in the serum of patients to be tested. It may also be possible to use a protein chip comprising an antibody according to the invention, this time to detect the presence of polypeptides in the serum of patients which are capable of being recognized by the said antibodies.

The subject of the present invention is also the use of a compound chosen from a nucleotide sequence, a polypeptide, a vector, a cell or an antibody according to the invention for the preparation of a medicament.

The pathologies more specifically targeted are viral diseases and diseases characterized by the development of tumour cells or cellular degeneration such as Alzheimer's disease or schizophrenia. Thus, the abovementioned medicament is intended for the prevention and/or the treatment of these diseases. In particular, the targeted disease is cancer.

One of the benefits of the present invention is that it has demonstrated the involvement of a large number of nucleotide sequences in the phenomena of tumour suppression, tumour reversion, apoptosis and/or viral resistance. These sequences are therefore expressed differentially when one of these abovementioned processes is triggered. Consequently, in the presence of a patient in whom the onset of one of these processes is suspected or for whom it is desired to check the absence of such an onset, it is useful to be able to determine, or even quantify, the expression of one or more sequences in accordance with the invention from a biological sample from the said patient. Optionally, analysis of the expression of one or more of the said sequences may be accompanied by a comparison with a reference level of expression corresponding to that of a healthy individual.

Consequently, the invention therefore also comprises a method for the diagnosis and/or prognostic evaluation of a viral disease or a disease characterized by tumour development or cell degeneration comprising the analysis of the expression of at least one sequence of the invention from a biological sample from a patient to be tested.

According to a preferred embodiment, the said method comprises the following steps:

-   -   isolation of the messenger RNA from a biological sample obtained         from a patient to be tested,     -   preparation of the complementary cDNA from the said messenger         RNA,     -   optionally, amplification of a portion of the complementary DNA         corresponding to at least one sequence of the invention, and     -   detection of the optionally amplified complementary DNA.

In particular, analysis of the expression of the sequence may be carried out by means of a DNA chip as described above.

Among the sequences of the present invention, some exhibit the characteristic of being receptors expressed at the surface of cells and in order to understand the mechanism thereof in the context of the abovementioned processes, it is useful to search for compounds capable of interacting with this receptor, that is to say of interacting with a polypeptide in accordance with the invention, it is also necessary to predict the secreted protein. This also applies to the polypeptides in accordance with the invention corresponding to secreted proteins (at the surface or outside the cells), hormone-like proteins, and the like. Consequently, the subject of the present invention is also a method for screening compounds capable of binding to a peptide in accordance with the invention and comprising the following steps:

-   -   bringing a polypeptide or a cell according to the invention into         contact with a candidate compound, and     -   detection of the formation of a complex between the said         candidate compound and the said polypeptide or the said cell.

A method for screening compounds may also be useful in relation to compounds capable of interacting with a nucleotide sequence according to the invention, or even with the sequences necessary for the expression or the regulation of these sequences. Indeed, the compounds are capable of interacting with the said sequences, the effect being to reduce, inhibit or on the contrary potentiate the expression of the sequences in question. Such a method comprises the following steps:

-   -   bringing a nucleotide sequence or a cell according to the         invention into contact with a candidate compound, and     -   detection of the formation of a complex between the said         candidate compound and the said nucleotide sequence or the said         cell.

For reasons mentioned above, it may therefore be useful to search for and/or assay, in a biological specimen or sample from a patient to be tested, the presence of a nucleotide sequence according to the invention. Such a method of detection and/or assay comprises the following steps:

-   -   bringing a labelled nucleotide sequence according to the         invention into contact with the biological sample to be tested,         under the conditions necessary for the formation of a hybrid,         and     -   detection and/or assay of the hybrid which may be formed between         the said nucleotide sequence and the nucleic acid present in the         said biological sample.

This method may additionally comprise a step for amplifying the nucleic acid of the said biological sample with the aid of primers chosen from the nucleotide sequences according to the invention.

In particular, this method may be carried out by means of the DNA chip described above.

Persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising:

-   -   a) a nucleotide sequence according to the invention, used as a         probe,     -   b) the reagents necessary for carrying out a hybridization         reaction between the said probe and the nucleic acid of the         biological sample,     -   c) the reagents necessary for the detection and/or assay of the         hybrid formed between the said probe and the nucleic acid of the         biological sample.

Such a kit may also contain positive or negative controls in order to ensure the quality of the results obtained.

Likewise, the detection and/or the assay of a polypeptide according to the invention can also be envisaged in the context of the present invention and this method therefore comprises the following steps:

-   -   bringing the biological sample into contact with a labelled         antibody according to the invention, and     -   detection and/or assay of the complex formed by the said         antibody of polypeptide present in the said sample.

Advantageously, this method may be carried out by means of the protein chip as described above. Here again, persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising:

-   -   a) a monoclonal or polyclonal antibody according to the         invention;     -   b) optionally reagents for preparing a medium favourable for the         antigen/antibody reaction;     -   c) the reagents allowing the detection of the antigen/antibody         complex.

Finally, the subject of the present invention is also a computer readable support or a computer support on which are recorded at least one nucleotide sequence according to the Claim 1 and/or at least one polypeptide sequence as defined in Claim 3 or 4. In particular, this support is chosen from the group comprising:

-   -   a) a floppy disk,     -   b) a hard disk,     -   c) a random access memory (RAM),     -   d) a read only memory (ROM),     -   e) a CD ROM.

The invention is not limited to the present description but encompasses on the contrary all the variants and will be understood more clearly in the light of the experimental data below.

FIGURES

FIG. 1 represents a curve for tumour growth for the U937 and US4 groups in SCID mice.

FIG. 2 represents a curve for the body weight of mice carrying a U937 or US4 tumour.

FIG. 3 illustrates:

a/ the analysis of the expression of the TPT1/TCTP gene in the U937/US4.2 lines and in the MCF7/MCF7+SIAH1 lines by Northern blotting. The quantities of total transcripts are checked by monitoring GAPDH;

b/ Western blot analysis of the expression of the TCTP protein in the U937/US4.2 model, in the MCF7/MCF7+siah model and analysis of the expression of the murine homologue of TCTP, TCTP_MOUSE, in the M1/LTR6 system after 20 h of induction at 32° C.;

c/ the analysis of the expression of TCTP in U937 cells transfected with the antisense TPT1/TCTP. The clones I and III are compared with the control, U937 cells with the vector alone.

On the right hand panel, detection of the cleavage of PARP, a marker for apoptosis, in the clones I and III, U937 cells transfected with the antisense TPT1/TCTP. Jurkat cells treated with an anti-Fas (anti-CD95) antibody, an inducer of apoptosis, serve as a control;

d/ characterization of the apoptotic cells by double labelling FITC-Annexin V and propidium iodide (PI).

The U937 cells+control vector and the U937 cells+antisense TPT1/TCTP are simultaneously labelled with annexin V coupled to FITC (which binds to the phosphatidylserine of the apoptotic cells in the early phase) and PI (which only labels necrotic cells whose plasma membrane is damaged). Cytofluorometric analysis makes it possible to determine the percentage of apoptotic cells (Annexin+, PI−), necrotic cells (Annexin+, PI+ or Annexin−, PI+) and intact cells (Annexin−, PI−);

e/ evaluation of apoptosis in the cells transfected with the antisense TPT1/TCTP, by in situ labelling of the free 3′ ends of the nuclear DNA: TUNEL (“Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling”) method. This labelling makes it possible to demonstrate in situ the fragmentation of nuclear DNA resulting from the activation of endonucleases during apoptosis. The apoptotic cells appear in green.

f/ the study of the in vivo tumorigenicity of the U937 cells transfected with: the vector alone (top curve—91 tumours/100), an antisense presenilin 1 (second curve from the top—12 tumours/20), SIAH1 (middle curve—12 tumours/20) and an antisense TPT1/TCTP, clone I and III (the bottom 2 curves—8 tumours/20 and 4 tumours/20). 10 million cells injected into each flank and into each shoulder.

FIG. 4 represents cells cultured on an extra-cellular tumour matrix (Matrigel). The 184B5 line, mammary epithelial cells transformed with benzo(a)pyrene, is used as a control for the formation of acini. The tumour cells T47D and MCF7 form irregular colonies. The MCF7 cells transfected with SIAH1 partially recover an organized structure. The MCF7 and T47D cells transfected with the antisense TPT1/TCTP result in a cellular reorganization comparable to the normal formation of the acini. The cells which appear green (light spots in the figure) are labelled with an anti-E_Cadherin antibody coupled to FITC and the nuclei labelled with PI appear red (darker spots).

Western blot analysis of the expression of TCTP in the MCF7 and T47D cells transfected with the antisense TPT1/TCTP.

1—DATA RELATING TO THE U937 and US4 CELLS

As seen above, the present invention uses the parental cells U937 and the “derived” cells US4. Indeed, the US4 cells and the US3 cells (which are not involved in the context of the present invention) share certain characteristics. Their mode of production and their properties are presented below.

Selection and Characterization of US3 and US4 Cells

The U937 cells were subjected to two series of limiting dilution until a single clonal population is obtained. These cells were infected with the H-1 parvovirus. The cytopathic effect of the virus creates massive cell death which spares two resistant clones which are US3 and US4 after three months of continuous culture. The survival of the cells is defined as the relative number of viable cells in the culture infected with the H-1 virus compared with the untreated culture, as measured four days after reinfection. To measure the tumorigenicity, 10⁷ U937, US3 and US4 cells were subcutaneously injected into scid/scid mice (4 or 5 weeks old). The tumorigenicity is expressed by the number of tumours developed by the mice within two months following the injection.

The approach was as follows: using clonal populations of malignant cells, subclones were derived with a suppressed tumorigenic phenotype. This selection made by means of the H-1 parvovirus is carried out by the elimination of the tumour cells which are preferentially killed while sparing the normal cells. The selection of the cells resistant to the cytopathic effect of the H-1 parvovirus outside a sensitive tumour can give rise to cells which have a reduced malignant phenotype.

On this basis, a clonal population of U937 cells was isolated, these cells are sensitive to the cytopathic effect of H-1 parvovirus and the US3 and US4 clones are, for their part, resistant to the virus. The US3 and US4 clones have a strong suppressed tumorigenic phenotype while the parental U937 cells develop tumours in 80% of cases among the scid/scid mice which were infected with the parvovirus, the US3 cells form only a single tumour and the US4 cells develop one tumour per 20 inoculations with 10⁷ cells. The results are assembled in the table below.

Resistance to the H-1 Parvovirus and Tumorigenicity of the U397, US3 and US4 Cells

Survival of the Tumorigenicity cells to in infection with the scid/scid Cell lines the H-1 virus mice U937 0.4 16/20 US3 96  0/10 US4 89  1/20

2—MATERIALS AND METHODS

The aim is to compare the growth of subcutaneous tumours in SCID mice induced by subcutaneous injection of transfected U937 and US4 human leukaemic cell lines.

A. Leukaemic Cell Lines and Culture Conditions

All the injected cells of the U937 (ATCC) and US4 cell lines were provided in the form of cell suspensions in bottles supplemented with RPMI-1640 culture medium supplemented with 2 mM L-glutamine, 10% foetal calf serum and gentamycin.

The U937 cell line is a CD4+ human monocytic cell line derived from a patient having a diffuse histiocytic lymphoma (1).

The cells were counted in a haemocytometer and their viability was tested with exclusions of trypan blue dyes at 0.25%. The viability was 95.5% and 90.5% for the U937 cells and the US4 cells, respectively. The U937 and US4 cells were centrifuged and then resuspended in an RPMI medium before being injected into SCID mice.

B. The animals

10 female SCID mice in good health (CB17/IcrHsd), 31 weeks old and weighing between 20 and 25 g, were supplied by Harlan France (Gannat, France). The animals were observed for 7 days in a specific room belonging to the Applicant which is the unit for treating specific-pathogen-free animals before their treatment. The unit for treating animals (INRA, Dijon, France) is authorized by the French Ministers of Agriculture and Research (authorization No. A21100). The animal experiments are carried out according to the European Ethical Directives for the well-being of animals in the context of experimental neoplasia (3).

B.1. Environment

The animals were kept in rooms under controlled conditions of temperature (24±1° C.), humidity (55±1%), light period (12 h of light/12 h of darkness) and air renewal. The animals were kept under SPF conditions and the temperature and the humidity of the room were continuously monitored. The aeration system was programmed to give rise to 14 air renewals per hour without recirculation. Fresh air from outside passes inside a series of filters before being uniformly diffused into each room. A high pressure (2 mm) was maintained in the experimentation rooms in order to prevent contamination or diffusion of pathogens within a mouse colony. All the staff working under SPF conditions followed specific directives as regards hygiene and clothing when they were in the breeding area.

B.2. Breeding

The animals were housed in polycarbonate cages (UAR, Epinay sur Orge, France) which were equipped so as to provide them with food and water. The standard size of the cages used is 637 cm² for 10 mice according to the standard internal operating procedures. The litter for animals and made up of sterile wood shavings (UAR) and is replaced twice per week.

B.3. Food and Drink

The animal food was bought from Extralabo (Provins, France). The food was provided ad libitum and was placed on the metal lid at the top of the cage. Water was also provided ad libitum from bottles of water equipped with rubber taps. The water bottles were washed, sterilized and replaced once per week. The water supply was sterilized by filtration with a 0.2 μm absolute filter.

B.4. Identification of the Animal and the Cage

After random distribution, the animals were identified with 2 different numbers engraved on the two ears. Each cage was marked with a specific code.

C—Experimental Data and Treatments

C.1. Induction of Tumours in SCID Mice

Before cell injection, the SCID mice were randomly divided into two groups, in an amount of 5 mice per group. 10⁷ US4 or U937 tumour cells in 0.2 ml of RPMI medium were subcutaneously inoculated at time 0 for each site of injection into SCID mice. Each animal received 4 injections of tumour cells located in different areas, one in each flank and one in each shoulder.

C.2. Collection of the Tumours

When the tumours reached a volume of 1 500 mm³, the mice were killed and the tumours were collected, weighed and frozen in liquid nitrogen, stored at −80° C. and then specifically labelled.

C.3. Monitoring of the Mice

Isoflurane forene (Minerve, Bondouble, France) was used to anaesthetize the animals before injection of cells for sacrificing. After injection of tumour cells, the mice were observed for 5 hours. The viability, behaviour, body weight of the mice and the growth of the subcutaneous tumour were recorded twice per week.

During the period of experimentation, the animals were killed under anaesthetic with isoflurane by cervical dislocation if one of the following signs appears:

-   -   sign of suffering (cachexia, weakness, difficulty in moving or         eating),     -   tumour growth up to 10% of the body weight,     -   tumour ulceration and persistently becoming nude,     -   position of the tumour interfering with movement and/or feeding,     -   loss of weight of 20% for three consecutive days.

An autopsy was performed on each animal to detect the possible presence of metastases or morphological abnormalities.

D—Presentation of the Data

D.1. Monitoring Parameters

The calculation for the median and mean survival time was expressed as follows: Mean survival time=S ₁/(S ₂ −NT) With:

-   -   S1=sum of the daily survivors from day 0 up to the end of the         experiment (without the survivors “not taken into account”*)     -   S2=number of animals at the beginning     -   NT=number of animals “not taken into consideration”*         *“not taken into consideration”: they are animals with tumours         which are smaller than the predetermined limit considered as         resulting from a defect in implantation of the tumour.

D.2. System for Inhibition of the Tumour

The tumour size was measured twice per week with a compass (and the tumour volume in mm³) will be estimated according to the formula: (length×width²/2 (4). The experiments were stopped when the tumour sizes in the mice reached 1 500 mm³.

-   -   After sacrificing, the tumours were excised and weighed.     -   The curve for tumour growth for the US4 and U937 groups was         noted using the mean of the tumour volumes.     -   The tumour doubling time for the US4 and U937 groups was defined         as the period required to reach a mean tumour volume of 200%         during the growth period.     -   The specific growth period over one or two doubling times (DT)         from injections of cells is defined as follows:         specific growth period=(DT US4−DT U937)/DT U937.     -   The growth period is calculated as the difference between the         median growth time for the US4 group and the U937 group in order         to reach the same tumour size.

D.3. Statistical Tests

All the statistical analyses were carried out with the StatView® software (Abacus Concept, Berkeley, USA). The statistical analyses of the changes in mean body weight, the tumour doubling time and the time to reach “V” were performed using the Bonferroni/Dunn test. A value p<0.05 was considered to be significant. All the groups were compared with each other.

E—Results

The curves for mean tumour volumes and body weights are shown in FIGS. 1 and 2 respectively.

No significant loss of body weight in SCID mice from the two groups was observed between day 8 and day 19.

A significant difference is observed as regards the time to reach “V” between the 2 groups of SCID mice whereas no significant difference was observed for the change in mean body weight (day 19−day 8) and for the doubling time.

The autopsies performed did not show the presence of metastases or of developments of suspicious nodosity. The tumours were collected on animals sacrificed after anaesthetic and cervical dislocation. The tumours were immediately placed in tubes, frozen in liquid nitrogen and stored at −80° C. The excised tumours had an ovoid shape, a moderate consistency and a pinkish colour. The interactions of the tumour with its environment (skin and muscle tissue) were limited and superficial.

F—Conclusions

The US4 cell line showed an uptake level significantly lower compared with the U937 cell line in the SCID mice.

The retardation of growth between the US4 and U937 tumours was 23.5 days and the doubling time was equivalent.

3—INVOLVEMENT OF TPT1/TCPT IN TUMOUR REVERSION

In order to study the involvement of TPT1/TCPT (Translationally controlled Tumour Protein encoding the Histamine Release Factor) in tumour reversion, the expression of the gene (FIG. 3 a) and the expression of the protein (FIG. 3 b) in various cellular models of tumour reversion: U937/US4.2, MCF7/MCF7+SIAH1 and M1/LTR6, were analysed. The level of expression of TPT1/TCTP is highly reduced in the revertant cells compared with the tumour cells.

To deal with the physiological aspect of this reduction, the U937 tumour cells were transfected with the antisense TPT1/TCTP. After having checked the drop in the expression of TPT1/TCTP (FIG. 3 c, left panel), the inventors detected its consequences in apoptosis by three different methods: detection of the cleavage of PARP, labelling with Annexin V and TUNEL method (FIG. 3 c, right panel; FIG. 3 d; FIG. 3 e, respectively).

The reduction in the expression of TPT1/TCTP in tumour cells in culture increases apoptosis.

The inventors then expressed in vivo the physiological consequences of this loss of TPT1/TCTP functions. We injected U937 cells transfected with antisense TPT1/TCTP into mice and observed a drastic reduction in the number of tumours formed when the expression of TPT1/TCTP is reduced (FIG. 3 f).

In parallel with these results, the reduction of TPT1/TCTP, obtained by transfection of double-stranded RNA specific for TPT1/TCTP (siRNA), into MCF7 and T47D cells, cultured on an extracellular matrix, leads to a cellular reorganization of acini which is comparable to a normal structure (FIG. 4).

REFERENCES

-   (1) Sundström C. and Nilsson K., Int. J. Cancer, 17, 565, 1976. -   (2) Principe d'éthique de l'expérimentation animale [Principle of     ethics of animal experimentation], Directive No. 86/609 EEC of 24     Nov. 1986, Decree No. 87/848 of 19 Oct. 1987, Application Order of     19 Apr. 1988. -   (3) United Kingdom co-ordinating committee on cancer research     guidelines for welfare of animals in experimental neoplasia, Br. J.     Cancer, 77: 1-10, 1998. -   (4) Bissery M. C. et al., Bull. Cancer, 78: 587, 1991. -   (5) Perricaudet et al. (1991). La Recherche 23: 471. -   (5a) Epstein (1992), Médecine/Sciences, 8, 902. -   (6) Temin (1986), Retrovirus vectors for gene transfer. In     Kucherlapati R., ed. Gene. -   (7) Carter (1993), Curr. Op. Biotechnology 3, 533. -   (8) Olins and Lee (1993), Curr. Op. Biotechnology 4: 520. -   (9) Buckholz (1993), Curr. Op. Biotechnology 4, 538. -   (10) Edwards and Aruffo (1993), Curr. Op. Biotechnology 4, 558. -   (11) Luckow (1993), Curr. Op. Biotechnology 4, 564. -   (11a) Rolfs, A. et al. (1991), Berlin: Springer-Verlag. -   (12) Walker (1992), Nucleic Acids Res. 20: 1691. -   (13) Kwoh et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 1173. -   (14) Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA, 87, 1874. -   (15) Kievitis et al. (1991), J. Virol. Methods, 35, 273. -   (16) Landegren et al. (1988) Science 241, 1077. -   (17) Segev (1992), Kessler C. Springer Verlag, Berlin, New York,     197-205. -   (18) Duck et al. (1990), Biotechniques, 9, 142. -   (19) Miele et al. (1983), J. Mol. Biol., 171, 281. -   (20) Matthews et al. (1988), Anal. Biochem., 169, 1-25. -   (21) Stewart and Yound (1984), Solid phase peptides synthesis,     Pierce Chem. Company, Rockford, 111, 2nd ed. (1984). 

1. An isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ. ID NO. 72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence having a percentage identity of al least 80%, after optimal alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d). 2-30. (canceled) 